Automated and robust method for recording nm-resolution 3d image data from serial ultra-sections of life-sciences samples with electron microscopes

Abstract

Methods of aligning specimen images of specimen sections situated on a substrate include obtaining an optical or SEM image of the substrate and locating and aligning optical or SEM images of each specimen section. The specimen sections are then imaged with an SEM to obtain preview images, and a region of interest (ROI) in at least one of the preview images is selected. The preview images are processed so that at least portions of the preview images proximate the ROI are aligned. Based on the alignment of the preview images, final SEM image of selected specimen sections are obtained so that a set of images aligned in three dimensions is available. Image alignment can use cross-correlation with a fixed or variable reference that can be updated as specimen section images are processed.

We claim: 
     
         1 . A method of obtaining a three-dimensional image of a specimen, comprising:
 sectioning the specimen into a plurality of specimen sections, wherein the specimen sections are obtained by slicing the specimen in a direction perpendicular to an axis through the specimen;   placing the specimen sections in order of the sectioning on a substrate;   with a camera, obtaining an overview image that includes a plurality of specimen section images on the substrate;   selecting a specimen section image in the overview image as a section template;   with a processor, locating each specimen section image based on the section template and assigning coordinates to each specimen section image;   with an electron microscope, obtaining preview images for a selected set of the plurality of specimen section images at a first resolution, the specimen section images of the selected set including a region of interest (ROI);   with the processor, determining an alignment of the preview images by tracking an image feature in the specimen section images based on at least one search template;   with the electron microscope, obtaining ROI images for the selected set of the plurality of specimen section images at a second resolution that is greater than the first resolution; and combining the ROI images based on the determined alignment to produce a three-dimensional reconstruction of the specimen.   
     
     
         2 . The method of  claim 1 , wherein the substrate is a silicon wafer. 
     
     
         3 . The method of  claim 1 , wherein the substrate is a tape strip. 
     
     
         4 . The method of  claim 1 , wherein the substrate is a plurality of tape strips or a plurality of silicon wafers, and the overview image includes images of the plurality of specimen sections on the plurality of silicon wafers or the plurality of tape strips. 
     
     
         5 . The method of  claim 1 , further wherein each of the preview images is aligned based on a reference preview image selected from the preview images. 
     
     
         6 . The method of  claim 5 , wherein each of the preview images is aligned based on a reference preview image selected as the preview image associated with an adjacent specimen section. 
     
     
         7 . The method of  claim 5 , wherein each preview image is aligned based on a correlation of a search template selected from the preview images. 
     
     
         8 . The method of  claim 1 , wherein the preview images include preview images 0, . . . , N, wherein N is an integer, and at least one preview image is aligned based on a search template selected from the preview images. 
     
     
         9 . The method of  claim 6 , wherein an i th  preview image is aligned by comparison with an (i−1) th  preview image, wherein i is an integer greater than one and less than N. 
     
     
         10 . The method of  claim 1 , further comprising processing the preview images to determine image transformations associated with alignment. 
     
     
         11 . The method of  claim 1 , further comprising determining stage coordinates associated with alignment of image areas associated with the specimen sections. 
     
     
         12 . An apparatus, comprising:
 a camera situated to obtaining an overview image that includes specimen section images for a plurality of specimen sections placed on a substrate on the substrate;   a processor operable to:   select a specimen section image in the overview image as a section template;   locate each specimen section image based on the section template and assign coordinates to each specimen section image; and   an electron microscope configured to obtain preview images for a selected set of the plurality of specimen section images at a first resolution, the specimen section images of the selected set including a region of interest (ROI), wherein the processor is further configured to, determine an alignment of the preview images by tracking an image feature in the specimen section images based on at least one search template, wherein the electron microscope is further situated to obtaining ROI images for the selected set of the plurality of specimen section images at a second resolution that is greater than the first resolution and the processor is configured to combine the ROI images based on the determined alignment to produce a three-dimensional reconstruction of the specimen.   
     
     
         13 . The apparatus of  claim 12 , wherein the processor is configured to align each of the preview images based on a reference preview image selected from the preview images. 
     
     
         14 . The apparatus of  claim 13 , wherein each of the preview images is aligned based on a reference preview image selected as the preview image associated with an adjacent specimen section. 
     
     
         15 . The apparatus of  claim 13 , wherein each preview image is aligned based on a correlation of a search template selected from the preview images. 
     
     
         16 . The apparatus of  claim 12 , wherein the preview images include preview images 0, . . . , N, wherein N is an integer, and at least one preview image is aligned based on a search template selected from the preview images. 
     
     
         17 . The apparatus of  claim 16 , wherein an i th  preview image is aligned by comparison with an (i−1) th  preview image, wherein i is an integer greater than one and less than N. 
     
     
         18 . The apparatus of  claim 15 , further comprising processing the preview images to determine image transformations associated with alignment. 
     
     
         19 . The apparatus of  claim 12 , wherein the processor is configured to determine stage coordinates associated with alignment of image areas associated with the specimen sections. 
     
     
         20 . The apparatus of  claim 19 , wherein the substrate is a tape strip.